CN88101007A - 血细胞分离 - Google Patents
血细胞分离 Download PDFInfo
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- CN88101007A CN88101007A CN198888101007A CN88101007A CN88101007A CN 88101007 A CN88101007 A CN 88101007A CN 198888101007 A CN198888101007 A CN 198888101007A CN 88101007 A CN88101007 A CN 88101007A CN 88101007 A CN88101007 A CN 88101007A
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- hydroxy alkyl
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
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- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3693—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits using separation based on different densities of components, e.g. centrifuging
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3693—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits using separation based on different densities of components, e.g. centrifuging
- A61M1/3695—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits using separation based on different densities of components, e.g. centrifuging with sedimentation by gravity
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- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
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Abstract
羟基烷基纤维素可用作无损伤性分离红细胞和白细胞的沉降剂。完整的WBC的回收率高于传统的方法。回收的白细胞可用于干扰素生产。
Description
本发明涉及在通常重力下,利用羟基烷基纤维素分离白细胞和红细胞的方法。所说的白细胞可用于生产干扰素。
通常采用羟基乙基淀粉做沉降剂。参见Lionetti,US.4,004,975;Pestka,US.4,289,690;Djerassi,US.4,111,199;Chadha,US.4,485,038;以及Van Oss,等人免疫学通风,10(6):549-55(1981)。采用HES(羟基乙基淀粉)处理方法,可回收原始试样中白细胞的68%,而使用本方法可回收80%。虽然氯化氨溶胞作用法可回收90%的白细胞,但由于损失红细胞而不是令人满意的。
用于这种细胞分离的其它试剂有葡聚糖(葡萄糖聚合物)、蔗聚糖(蔗糖聚合物)、PVP、胡芦巴(fenugreek)种子提取液,以及植物血细胞凝集素。Lichtenstein,US.3,709,701;Chany,US.3,560,611;Ferrante,US.4,255,256;Guirgis,US.4,152,208;Furuta,US.4,409,106;Goore,US.3,800,035;Shepherd,US.3,594,276;Kirkham,US.3,635,798;Widmark,US.3,700,555。
Kanter,US.4,487,700利用一种中密度触变阻挡物质(thixotropic barrier material)从红细胞和吞噬白细胞中分离淋巴细胞。Meyst,US.4,283,289描述了一种白细胞过滤器。利用NH4Cl溶裂红细胞,而使大多数白细胞保持完整也是已知的。
羟基烷基纤维素,特别是羟基乙基纤维素,在药物和其它组合物中被用作增稠剂和稳定剂,但还从未用作细胞沉降剂。
本发明涉及利用羟基烷基纤维素,特别是羟基甲基纤维素(HMC)、羟基乙基纤维素(HEC)、羟基丙基纤维素(HPC)和羟基丁基甲基纤维素(HBMC)在细胞分离时作为沉降剂。使用本发明的方法,从上层相中,原始白细胞的80%可回收,并是完整的,95%以上存活,而且其下层相中的红细胞也是完整无损的。
由于HEC在混合物中的最终浓度为0.05%,与传统分离方法中3%的HES相比,最后回收的细胞上粘附很少的HEC,且容易洗掉。
与NH4Cl溶裂作用不同,HEC技术不会损坏粒细胞(颗粒白细胞)或红细胞。此外,用本技术回收的白血细胞暴露在诱发剂中,至少可贮存一天而不会损害α-干扰素的生产。
实例1
汇集血沉棕黄层(美国红十字会),混合并取样。用Cell Dyn 400型细胞计数仪(Sequoia International)测定原始的红血细胞(RBC)和白血细胞(WBC)数。沉降剂的制备方法如下:用去离子水配制0.1%HEC(W/V)和0.9%NaCl(W/V)的溶液,至少搅拌半小时直至HEC完全溶成溶液。将该沉降剂慢慢与血沉棕黄层试样混合至少一分钟,倒进一个分液漏斗或其它合适的容器中,于室温或更低温度下,放置1 1/2 ~3小时使其沉淀,HEC在该混合液中的最终浓度以0.05%(W/V)为最佳。但是,HEC的最终浓度可以在0.025~0.5%之间。分离完成后,底层的红细胞层被排放出或吸取顶层的白细胞层。底层可用前述方法用新的沉降剂再行沉降,以获取更多的白细胞。
对所收集的各层,测其RBC、WBC数,WBC/RBC,以及WBC的活力(采用锥虫蓝排斥试验)。结果如下:
实验1476-19(0.1%HEC,0.9%NaCl贮备液)
样品 体积 RBC×1012WBC×1010WBC/RBC 回收率
试样 600毫升 3.25 4.00 0.01 100%
顶层 720毫升 0.20 3.37 0.17 84%
底层 500毫升 2.73 0.34 0.001 9%
顶层(清
洗后) 160毫升 - 3.12 - 78%
实验1476-7(0.1%HEC,0.9%NaCl贮备液)
试样 1000毫升 5.88 4.66 0.01 100%
顶层 830毫升 0.11 3.72 0.34 80%
底层 1190毫升 5.45 1.09 0.002 23%
顶层(清
洗后) 130毫升 0.07 3.58 0.51 77%
采集后的细胞可用离心法清洗,或者用切向流动过滤法及其传统的方法。
所说的WBC层在1000×g下旋转7分钟,弃去上层清液,在37℃下将片状物重新悬浮在白细胞培养基中,并进行WBC计数。将WBC加到2L容积的MEM培养基、人血清和α-干扰素引物中,浓度为1×10°7WBC/毫升,在一个6L的置于37℃水浴中的园底烧瓶中进行诱发。最初保温3小时后,在所说烧瓶中加入Sendai病毒以诱发α-干扰素的生长。典型的诱发期为18小时。然后在4000g下离心20分钟,采集α-干扰素。将上清液的样品用于CPE(致细胞病效应)测定。
所说的CPE测定是基于α-干扰素具有保护某些细胞免受某些病毒侵害的能力。在所用的测定中,将Hep-2细胞培养在96槽的微型滴定板上。将一系列的α-干扰素稀释液加到这些试样槽上,并与这些细胞一起保温20小时(37℃,5%CO2)左右。接下来,槽内加入VSV病毒以感染未受保护的细胞。经过一段足够长的保温期,以至使对照槽(即仅存在所说的细胞和病毒的那些槽)中的细胞100%死之时,用龙胆染色剂对所有的槽染色,完整的细胞因为细胞膜着色而呈紫色。有50%细胞受到保护的槽的编号(即该槽的稀释度)称为终点。然后用该终点来将样品的滴定度(以干扰度/ml为单位)与由NIH得到的标准相关连。
当0.05%HEC的储备液用作细胞分离时,分离速度很慢。即与0.1%和0.2%HEC储备液进行的分离相比,在可比拟的时间段上,0.05%HEC的分离不如那两者。并且,所说的0.05%的分离似乎在中间层有较多的红细胞混杂(其中段为粉红色可证实这一点,而在其它两种情况为白色)。
0.5%的HEC储备液不能给出很好的分离-仅仅形成两层,而且从底层回收到比顶层更多的WBC。此外,顶层的“纯度”,用WBC/RBC表示时小于0.1%HEC分离的合并了的二个上层的1/2(即0.14对照0.30的关系)。0.7%和1.0%HEC的分离效果更差。
经过一系列实验,得到了20-50,000单位/ml的α-干扰素滴定度。
制备一个用HEC法收集的WBC的细胞自旋涂片(Cytospin Smear)。这些细胞用MAY-GRUNWALD-GIEMSA染色剂处理,在显微镜下观察。细胞鉴别研究表明,该白细胞亚群具有与原始血试样基本相同的分布,白细胞的形态正常。
实例2
用前述制备HEC溶液的方法制备0.1%羟基丙基纤维素溶液(300,000m.W.;Aldrich Chemical)。0.1%HPC/0.9%NCl溶液与相同体积的血沉棕黄色试样混合。在两个单独的实验1476-19C和1426-21中,观察到WBC和RBC很好的分离。可由于上述分离都不是在分液漏斗中进行的,所以收集顶层时,难免不混进处于底层的RBC。
用羟基丁基甲基纤维素(HBMC)作同类型的实验。用同样0.1%浓度的该聚合物和0.9%NaCl进行分离,分离似乎比同时进行的HEC的分离速度快,看来也更好。
通过一些方法可以提高WBC的回收率,首先,所说的沉降可在一个有利于RBC/WBC分离的器具中进行,像一个在预计的RBC/WBC界面位置上有一个收缩的器具,或带有设计成能尽量减小所说界面扰动的排出或吸出装置。第二,可以加入一种触变剂,该触变剂的比重使得它可以聚集成一个分离RBC和WBC层的阻档结构。第三,用未使用过的沉降剂按需要重复提取步骤。
除HEC和HPC外,其它的羟基烷基纤维素也可用作沉降剂。
HEC不仅可用于单位重力沉降法,还可用于离心法分离RBC/WBC。HEC还可用于从细胞残渣中分离细胞。由本技术提供的红细胞和白细胞可以被分离成WBC或RBC的亚型,或分为几部分,产生各种WBC和RBC的组份。
Claims (13)
1、一种将含红细胞和白细胞的血液或血液成分分离成一个富含白细胞的第一成分和一个富含红细胞的第二成分的方法,其特征在于将所说的血液或血液成分与一种含有羟基烷基纤维素的沉降剂混合,使该混合物沉降,并将该混合物分离成所说的第一和第二成份。
2、根据权利要求1的方法,其中所说的沉降在单位重力下发生。
3、根据权利要求1的方法,其中所述的羟基烷基纤维素在所说混合液中的最终浓度为0.025%至0.5%。
4、根据权利要求1的方法,其中所述的羟基烷基纤维素为羟基乙基纤维素。
5、根据权利要求1的方法,其中所述的羟基烷基纤维素是羟基丙基纤维素。
6、根据权利要求1的方法,其中在该富含白细胞成分中回收80%以上的原始白细胞。
7、根据权利要求1的方法,进一步包括使用一种触变性阻档物质来分离所说的第一和第二成分。
8、根据权利要求1的方法,细胞没有暴露于破坏红细胞和粒细胞的条件下。
9、根据权利要求1的方法,其中所述的羟基烷基纤维素为羟基甲基纤维素。
10、根据权利要求1的方法,其中所述的羟基烷基纤维素是羟基丁基甲基纤维素。
11、一种获得适用于诱发干扰素的白细胞的方法,其特征在于提供血液或含白血球的血液成分,将该血液与含有羟基烷基纤维素的沉降剂混合,取其中富含白细胞的上层,并从所说上层中回收适于诱发干扰素的白细胞。
12、根据权利要求11的方法,其中所述的白细胞在经过一天的贮存之后仍适于干扰素的诱发。
13、根据权利要求11的方法,其中所述的羟基烷基纤维素是从羟基甲基、羟基乙基、羟基丙基和羟基丁基甲基纤维素中选取的。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US07/003,838 US4744907A (en) | 1987-01-16 | 1987-01-16 | Blood cell separation |
US003838 | 1995-09-15 |
Publications (1)
Publication Number | Publication Date |
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CN88101007A true CN88101007A (zh) | 1988-09-07 |
Family
ID=21707830
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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CN198888101007A Pending CN88101007A (zh) | 1987-01-16 | 1988-01-15 | 血细胞分离 |
Country Status (11)
Country | Link |
---|---|
US (1) | US4744907A (zh) |
EP (1) | EP0276931B1 (zh) |
JP (1) | JPH02501890A (zh) |
CN (1) | CN88101007A (zh) |
AT (1) | ATE73665T1 (zh) |
AU (1) | AU1189888A (zh) |
CA (1) | CA1299104C (zh) |
DE (1) | DE3869146D1 (zh) |
IL (1) | IL85112A0 (zh) |
NZ (1) | NZ223221A (zh) |
WO (1) | WO1988005331A1 (zh) |
Families Citing this family (11)
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US6129930A (en) | 1993-09-20 | 2000-10-10 | Bova; David J. | Methods and sustained release nicotinic acid compositions for treating hyperlipidemia at night |
US20060263428A1 (en) * | 1993-09-20 | 2006-11-23 | Eugenio Cefali | Methods for treating hyperlipidemia with intermediate release nicotinic acid compositions having unique biopharmaceutical characteristics |
US6746691B2 (en) | 1993-09-20 | 2004-06-08 | Kos Pharmaceuticals, Inc. | Intermediate release nicotinic acid compositions for treating hyperlipidemia having unique biopharmaceutical characteristics |
US6676967B1 (en) * | 1993-09-20 | 2004-01-13 | Kos Pharmaceuticals, Inc. | Methods for reducing flushing in individuals being treated with nicotinic acid for hyperlipidemia |
US6818229B1 (en) | 1993-09-20 | 2004-11-16 | Kos Pharmaceuticals, Inc. | Intermediate release nicotinic acid compositions for treating hyperlipidemia |
US7169547B2 (en) | 1994-12-05 | 2007-01-30 | New York Blood Center, Inc. | High concentration white blood cells as a therapeutic product |
US5789147A (en) * | 1994-12-05 | 1998-08-04 | New York Blood Center, Inc. | Method for concentrating white cells from whole blood by adding a red cell sedimentation reagent to whole anticoagulated blood |
WO1996032178A1 (en) * | 1995-04-13 | 1996-10-17 | Travenol Laboratories (Israel) Ltd. | Leukocyte filtration method and apparatus |
US8304185B2 (en) | 2009-07-17 | 2012-11-06 | Canon U.S. Life Sciences, Inc. | Methods and systems for DNA isolation on a microfluidic device |
JP5795255B2 (ja) | 2008-07-18 | 2015-10-14 | キヤノン ユー.エス. ライフ サイエンシズ, インコーポレイテッドCanon U.S. Life Sciences, Inc. | 微小流体dna試料調製のための方法およびシステム |
CN101971796B (zh) * | 2010-05-26 | 2013-04-17 | 赛业(广州)生物科技有限公司 | 无蛋白非程序细胞冻存液 |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
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CA559303A (en) * | 1958-06-24 | E. Gloor Walter | Liquid clarification with coagulation | |
US3338828A (en) * | 1964-07-29 | 1967-08-29 | Joseph R Clark | Purification of water supplies and aqueous wastes |
DE1617401A1 (de) * | 1966-05-02 | 1972-02-17 | Centre Nat Rech Scient | Verfahren zur Herstellung von Interferon-Praeparaten |
US4004975A (en) * | 1975-12-30 | 1977-01-25 | The United States Of America As Represented By The Secretary Of The Navy | Method of isolating and cryopreserving human white cells from whole blood |
US4255256A (en) * | 1978-12-13 | 1981-03-10 | Antonio Ferrante | Medium for the separation of human blood leucocytes |
AU524397B2 (en) * | 1978-12-13 | 1982-09-16 | Antonio Ferrante | Medium for separation of human blood leukocytes |
DE3009126A1 (de) * | 1980-03-10 | 1981-10-08 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., 3400 Göttingen | Verfahren zur gewinnung von funktionell und morphologisch intakten und lebensfaehigen leukozyten aus blut |
US4487700A (en) * | 1983-02-18 | 1984-12-11 | Technicon Instruments Corporation | Method and apparatus for separating lymphocytes from anticoagulated blood |
GB8411192D0 (en) * | 1984-05-02 | 1984-06-06 | Celltech Ltd | Separating animal cells from liquid culture |
-
1987
- 1987-01-16 US US07/003,838 patent/US4744907A/en not_active Expired - Fee Related
-
1988
- 1988-01-11 AU AU11898/88A patent/AU1189888A/en not_active Abandoned
- 1988-01-11 WO PCT/US1988/000053 patent/WO1988005331A1/en unknown
- 1988-01-11 JP JP62506338A patent/JPH02501890A/ja active Pending
- 1988-01-15 IL IL85112A patent/IL85112A0/xx unknown
- 1988-01-15 CA CA000556674A patent/CA1299104C/en not_active Expired - Fee Related
- 1988-01-15 AT AT88300325T patent/ATE73665T1/de not_active IP Right Cessation
- 1988-01-15 DE DE8888300325T patent/DE3869146D1/de not_active Expired - Lifetime
- 1988-01-15 CN CN198888101007A patent/CN88101007A/zh active Pending
- 1988-01-15 EP EP88300325A patent/EP0276931B1/en not_active Expired - Lifetime
- 1988-01-18 NZ NZ223221A patent/NZ223221A/xx unknown
Also Published As
Publication number | Publication date |
---|---|
AU1189888A (en) | 1988-08-10 |
DE3869146D1 (de) | 1992-04-23 |
JPH02501890A (ja) | 1990-06-28 |
NZ223221A (en) | 1989-06-28 |
WO1988005331A1 (en) | 1988-07-28 |
EP0276931B1 (en) | 1992-03-18 |
ATE73665T1 (de) | 1992-04-15 |
IL85112A0 (en) | 1988-06-30 |
EP0276931A1 (en) | 1988-08-03 |
US4744907A (en) | 1988-05-17 |
CA1299104C (en) | 1992-04-21 |
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